SUMMARY Hematopoietic stem cells (HSCs) sustain life-long blood formation due to their ability to self-renew and differentiate into all mature blood cell types (referred to as ?stemness?). Transplantation of HSC-containing grafts is a life-saving therapy for multiple blood disorders; however, shortage of immunologically matched donors limits the number of patients that can be treated. Expansion/generation of human HSCs in culture would greatly improve transplantation therapy but has been unsuccessful due to poor understanding of the underlying biology of HSC stemness. We identified MLLT3 as HSC regulator that is highly enriched in human HSC at all stages once they have emerged from AGM and expand in the fetal liver (FL); however, MLLT3 expression declines in cultured HSC. Loss and gain of function studies on human FL and cord blood (CB) HSCs showed that MLLT3 is critical for their self-renewal, and when its expression is restored, it enables HSC expansion in culture by protecting their ?stemness? program. Importantly, FL and CB HSC expanded in culture showed 10-30 fold increase in human engraftment in NSG mice, and ability to sustain the HSPC compartment and multilineage hematopoiesis without malignant transformation or differentiation block. This finding offers an unprecedented opportunity to understand how human HSC stemness is controlled, and harness MLLT3 for clinical use. So far, the regulatory mechanisms that govern MLLT3 expression in HSC are completely unknown. Using a combination of epigenetic studies and bioinformatic approaches, we identified several candidate enhancers in MLLT3 gene that may regulate its two isoforms. Our data suggests that these enhancers are epigenetically remodeled during differentiation and silenced during HSC culture, providing new avenues to understand why cultured HSC lose MLLT3 expression. To understand how MLLT3 expression is regulated in human HSCs, we propose two complementary approaches: 1) CRISPR/Cas9-mediated epigenome editing to dissect the role of MLLT3 enhancers, 2) combination of bioinformatic and experimental approaches using lentiviral overexpression to identify MLLT3 upstream regulators. Success in these approaches could help generate/expand human HSCs in culture and thereby increase the availability of HSCs for transplantation.